Different effects of the TAR structure on HIV-1 and HIV-2 genomic RNA translation

Abstract

The 5'-untranslated region (5'-UTR) of the genomic RNA of human immunodeficiency viruses type-1 (HIV-1) and type-2 (HIV-2) is composed of highly structured RNA motifs essential for viral replication that are expected to interfere with Gag and Gag-Pol translation. Here, we have analyzed and compared the properties by which the viral 5'-UTR drives translation from the genomic RNA of both human immunodeficiency viruses. Our results showed that translation from the HIV-2 gRNA was very poor compared to that of HIV-1. This was rather due to the intrinsic structural motifs in their respective 5'-UTR without involvement of any viral protein. Further investigation pointed to a different role of TAR RNA, which was much inhibitory for HIV-2 translation. Altogether, these data highlight important structural and functional differences between these two human pathogens.

Figure 1.

Low HIV-2 Gag production in human cells. (A) Wild-type pNL4.3 (HIV-1) and pROD10 (HIV-2) proviral plasmids (upper scheme, regulatory and accessory proteins were omitted for simplicity) were transfected in HeLa cells and cells extracts from cells expressing HIV-1 and HIV-2 were prepared and probed against Gag by western blot as described in ‘Materials and Methods’ section. In parallel, viral production was determined by measuring the RT activity present in supernatant of transfected cells as described in ‘Materials and Methods’ section (left graph). (B) Schematic representation of the HIV-1 and HIV-2 proviral-Renilla DNA constructs used in this study (regulatory and accessory proteins were omitted for simplicity). They correspond to pNL4.3 (HIV-1) and pROD10 (HIV-2) proviral plasmids in which the Renilla reporter gene was inserted in frame within the Gag coding region as described in ‘Materials and Methods’ section. gRNA levels (left graph) and the translation efficiency from the gRNA (right graph) were determined in T-lymphocytes (black bars) and HeLa cells (gray bars) at 24 hpt as indicated. Results are normalized to values obtained for pNL4.3-Renilla (arbitrary set to 1) and expressed as mean ± SD corresponding to values obtained in three duplicate independent experiments.

Figure 2.

Translation of the HIV-2 gag mRNA is inefficient in cells. (A) About 0,125 pmoles of in vitro transcribed HIV-2 and HIV-1 gag mRNAs (see cartoon on the graph) were transfected into HeLa cells as described in ‘Materials and Methods’ section. Renilla activity (graph on the left) and transfected mRNA levels (graph on the right) were measured at 3 hpt. Results were normalized to values obtained for HIV-1 gag mRNA (HIV-1 Gag, arbitrary set to 1) and expressed as mean ± SD corresponding to values obtained in three independent duplicate experiments. (B) Specific activities of in vitro translated ³⁵S-labeled HIV-1 and HIV-2 Gag–Renilla fusion proteins were analyzed as described in ‘Materials and Methods’ section. Results were first normalized by the methionine content and then to values obtained for HIV-1 Gag–Renilla (HIV-1 Gag, arbitrary set to 1) and expressed as mean ± SD corresponding to values obtained in three independent experiments. (C) About 0.125 pmol of HIV-2 and HIV-1 gag mRNAs were transfected in HeLa cells and 3 hpt CHX was added at 100 µg/ml. Renilla activity was analyzed at 0, 30, 60 and 90 min post CHX treatment as indicated in the figure. Results are normalized to values obtained at time 0 (arbitrary set to 1) and expressed as mean ± SD corresponding to values obtained in three independent duplicate experiments. (D) About 0.125 pmol of HIV-2 and HIV-1 gag mRNAs were transfected in Cos7 cells, 3T3 cells or human macrophages or translated in vitro in the URRL system and Renilla activity was measured at 3 hpt (transfected cells) or 30 min (in vitro translation). Results were normalized to values obtained for HIV-1 gag mRNA (HIV-1 Gag, arbitrary set to 1) and expressed as mean ± SD corresponding to values obtained in three independent duplicate experiments. (E) About 0.125 pmol of HIV-2 and HIV-1 gag mRNAs were transfected in HeLa cells and Renilla activity was measured at 0, 1, 2, 3, 4 and 6 hpt. Results are normalized to values obtained for HIV-1 gag mRNA (arbitrary set to 1 at 6 hpt point time) and expressed as mean ± SD corresponding to values obtained in three independent duplicated experiments. (F) About 0.125 pmol of gag mRNAs derived from different HIV-2 strains were transfected in HeLa cells and Gag–Renilla expression was compared to HIV-1. Renilla activity was measured at 3 hpt. Results are normalized to values obtained for HIV-1 (arbitrary set to 1) and expressed as mean ± SD corresponding to values obtained in three independent duplicate experiments.

Figure 3.

The HIV-2 5′-UTR impairs Gag expression. About 0.125 pmol of chimeric gag mRNAs in which the 5′-UTR of one virus was appended to the Gag coding region of the other (see cartoon) were transfected in HeLa cells and Renilla activity was measured at 3 hpt. In the cartoon, HIV-2/HIV-1 Gag refers to the RNA that harbors the HIV-2 5′-UTR followed by the HIV-1 Gag coding region. HIV-1/HIV-2 RNA refers to the Renilla RNA that harbors the HIV-1 5′-UTR followed by the HIV-2 Gag coding region. Results were normalized to values obtained for wild-type HIV-1 gag mRNA (HIV-1 Gag, arbitrary set to 1) and expressed as mean ± SD corresponding to values obtained in three independent duplicate experiments.

Figure 4.

The HIV-2 5′-UTR interferes with ribosome recruitment. (A) About 0.125 pmol of Renilla RNAs in which translation was driven by different 5′-UTR (see table on the left) were transfected in HeLa cells as described in ‘Materials and Methods’ section. Renilla activity was measured at 3 hpt. Results were normalized to values obtained for human β-globin 5′-UTR RNA (arbitrary set to 1) and expressed as mean ± SD corresponding to values obtained in three independent duplicate experiments. (B) About 0.125 pmol of 5′-UTR-Renilla RNAs (see cartoon on the graph) were transfected in HeLa cells. Renilla activity and transfected RNA levels were determined at 3 hpt as described in ‘Materials and Methods’ section. Results were normalized to values obtained for HIV-1 5′-UTR RNA (arbitrary set to 1) and expressed as mean ± SD corresponding to values obtained in three independent duplicate experiments. (C) About 0.125 pmol of 5′-UTR-Renilla RNAs (see cartoon on the graph) were transfected in Cos7 and 3T3 cells or human macrophages as described in ‘Materials and Methods’ section. Renilla activity was measured at 3 hpt. Results were normalized to values obtained for HIV-1 5′-UTR RNA (arbitrary set to 1) and expressed as mean ± SD corresponding to values obtained in three independent duplicate experiments. (D) About 0.125 pmol of Renilla mRNAs containing the 5′-UTR derived from different HIV-2 strains were transfected in HeLa cells and Renilla expression was compared to HIV-1. Renilla activity was measured at 3 hpt. Results were normalized to values obtained for HIV-1 5′-UTR RNA (arbitrary set to 1) and expressed as mean ± SD corresponding to values obtained in three independent duplicate experiments. (E) About 0.125 pmol of Renilla RNAs in which translation was driven by the 5′-UTR of different naturally occurring HIV-1 isolates (indicated as Pat#4, 8, 15, 18, 21) together with β-globin, HIV-1 NL4-3 and HIV-2 Rod10 (used as control references) were transfected in HeLa cells as described in ‘Materials and Methods’ section. Renilla activity was measured at 3 hpt. Results were normalized to values obtained for human β-globin 5′-UTR RNA (arbitrary set to 1) and expressed as mean ± SD corresponding to values obtained in three independent duplicate experiments.

Figure 5.

TAR interferes with ribosome recruitment onto the HIV-2 gRNA. (A) About 0.125 pmol of Renilla mRNAs containing the wild-type viral 5′-UTR or 5′ and 3′ deletions (see cartoon on the graph) were transfected in HeLa as described in ‘Materials and Methods’ section. Renilla activity was measured at 3 hpt. Results were normalized to values obtained for HIV-1 5′-UTR RNA (arbitrary set to 1) and expressed as mean ± SD corresponding to values obtained in three independent duplicate experiments. (B) Secondary structure prediction and stability of HIV-1 and HIV-2 TAR RNA structure using the mfold software as indicated in ‘Materials and Methods’ section. (C) The untreated rabbit reticulocyte lysate was programmed with 0.125 pmol of wild-type or ΔTAR Renilla mRNAs and Renilla activity was measured after 30 min. Results were normalized to values obtained for HIV-1 5′-UTR RNA (arbitrary set to 1) and expressed as mean ± SD corresponding to values obtained at least in three independent experiments. (D) The untreated rabbit reticulocyte lysate was programmed with 0.125 pmol of wild-type or ΔTAR Renilla mRNAs in the presence of buffer or HeLa cells extracts as described in ‘Materials and Methods’ section. Renilla activity was measured after 30 min. Results were normalized to values obtained for HIV-1 5′-UTR RNA in the presence of buffer (arbitrary set to 1) and expressed as mean ± SD corresponding to values obtained at least in three independent experiments.

Figure 6.

HIV-2 TAR stability impairs ribosome recruitment. (A) HeLa cells were transfected with equal molar quantities of Renilla mRNAs as indicated in the figure and cells extracts were prepared at 3 hpt for western blot analyses. In parallel, cells extracts from mock transfected cells or mock transfected cells treated with sodium arsenite during 30 min were also prepared and used as controls. (B) HeLa cells were transfected with 0.125 pmol of a control RNA or with HIV-1 5′-UTR, HIV-2 5′-UTR or HIV-2 ΔTAR-Renilla RNAs and whole cellular extracts were prepared at 3 hpt and probed against eIF2α, eIF2α phosphorylated and PABP by western blot as described in ‘Materials and Methods’ section. (C) Schematic representation not at scale of the HIV-2 5′-UTR indicating the position of the alternative splice donor (SD′) and splice acceptor (SA′) that results in the removal of the 5′-UTR intron described by Lodmell et al. (67). About 0.125 pmol of wild-type or 5′-UTR spliced HIV-2 Renilla mRNA (left graph) or 5′-UTR spliced HIV-2 Gag–Renilla mRNA (right graph) were transfected in HeLa cells together with the corresponding HIV-1 Renilla mRNA and Renilla activity was measured at 3 hpt. Results were normalized to values obtained for HIV-1 mRNAs (arbitrary set to 1) and expressed as mean ± SD corresponding to values obtained in three independent duplicate experiments.